Metabolic network reconstruction and genome-scale model of butanol-producing strain Clostridium beijerinckii NCIMB 8052

<p>Abstract</p> <p>Background</p> <p>Solventogenic clostridia offer a sustainable alternative to petroleum-based production of butanol--an important chemical feedstock and potential fuel additive or replacement. <it>C. beijerinckii </it>is an attractive microorganism for strain design to improve butanol production because it (i) naturally produces the highest recorded butanol concentrations as a byproduct of fermentation; and (ii) can co-ferment pentose and hexose sugars (the primary products from lignocellulosic hydrolysis). Interrogating <it>C. beijerinckii </it>metabolism from a systems viewpoint using constraint-based modeling allows for simulation of the global effect of genetic modifications.</p> <p>Results</p> <p>We present the first genome-scale metabolic model (<it>i</it>CM925) for <it>C. beijerinckii</it>, containing 925 genes, 938 reactions, and 881 metabolites. To build the model we employed a semi-automated procedure that integrated genome annotation information from KEGG, BioCyc, and The SEED, and utilized computational algorithms with manual curation to improve model completeness. Interestingly, we found only a 34% overlap in reactions collected from the three databases--highlighting the importance of evaluating the predictive accuracy of the resulting genome-scale model. To validate <it>i</it>CM925, we conducted fermentation experiments using the NCIMB 8052 strain, and evaluated the ability of the model to simulate measured substrate uptake and product production rates. Experimentally observed fermentation profiles were found to lie within the solution space of the model; however, under an optimal growth objective, additional constraints were needed to reproduce the observed profiles--suggesting the existence of selective pressures other than optimal growth. Notably, a significantly enriched fraction of actively utilized reactions in simulations--constrained to reflect experimental rates--originated from the set of reactions that overlapped between all three databases (<it>P </it>= 3.52 × 10^-9, Fisher's exact test). Inhibition of the hydrogenase reaction was found to have a strong effect on butanol formation--as experimentally observed.</p> <p>Conclusions</p> <p>Microbial production of butanol by <it>C. beijerinckii </it>offers a promising, sustainable, method for generation of this important chemical and potential biofuel. <it>i</it>CM925 is a predictive model that can accurately reproduce physiological behavior and provide insight into the underlying mechanisms of microbial butanol production. As such, the model will be instrumental in efforts to better understand, and metabolically engineer, this microorganism for improved butanol production.</p

Chitin hydrolysis and N-acetylglucosamine utilization by solventogenic clostridia

Environment pollution and energy supply are among the huge problems which threaten the world, especially in industrialised countries. Several studies have considered how to exploit waste materials as renewable substrates for various industries to obtain different products. Some wastes from the aquatic food industry contain a considerable amount of the N-acetylglucosamine (NAG) polymer chitin, which has potential as a substrate for the solventogenic clostridia in the acetone-butanol-ethanol fermentation. Development of an effective process will, however, depend on a detailed understanding of the mechanism and control of chitin hydrolysis and NAG metabolism. Clostridium beijerinckii NCIMB 8052 was shown to exhibit chitinase activity and to be able to grown on NAG. The predominant mechanism for uptake of sugars and sugar derivatives in the clostridia is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). Extracts of C.beijerinckii grown on NAG exhibited a phosphotransferase activity for NAG which was also present in extracts of cells grown on glucose, consistent with the observation that glucose did not repress utilization of NAG in media containing both substrates. Genomic analysis has identified genes encoding a putative nag-pts that belongs to the glucose family of PTS permeases. Two divergent genes encode the IIA and IICB domains of the PTS, and are associated with a gene encoding a putative transcriptional antiterminator. These genes were found to be expressed in cells growing on NAG or glucose, but not glucitol. The role of the putative nag-pts genes in NAG uptake was confirmed by functional analysis. An artificial NAG operon was constructed in which the nag-pts genes were in series and expression of the operon in Escherichia coli mutants provided evidence for the ability of the PTS to transport and phosphorylate NAG and glucose, but not mannose

TargeTron technology applicable in solventogenic clostridia: Revisiting 12 years’ advances

Clostridium has great potential in industrial application and medical research. But low DNA repair capacity and plasmids transformation efficiency severely delayed development and application of genetic tools based on homologous recombination (HR). TargeTron is a gene editing technique dependent on the mobility of group II introns, rather than homologous recombination, which made it very suitable for gene disruption of Clostridium. The application of TargeTron technology in Clostridium was academically reported in 2007 and this tool has been introduced in various clostridia as it is easy to operate, time-saving, and reliable. TargeTron has made great progress in solventogenic Clostridium in the aspects of acetone-butanol-ethanol (ABE) fermentation pathway modification, important functional genes identification, and xylose metabolic pathway analysis & reconstruction. In the review, we revisited 12 years' advances of TargeTron technology applicable in solventogenic Clostridium, including its principle, technical characteristics, application and efforts to expand its capabilities, or to avoid potential drawbacks. Some other technologies as putative competitors or collaborators are also discussed. We believe that TargeTron combined with CRISPR/Cas-assisted gene/base editing and gene-expression regulation system will make a better future for clostridial genetic modification

Mathematical modelling of clostridial acetone-butanol-ethanol fermentation

Clostridial acetone-butanol-ethanol (ABE) fermentation features a remarkable shift in the cellular metabolic activity from acid formation, acidogenesis, to the production of industrial-relevant solvents, solventogensis. In recent decades, mathematical models have been employed to elucidate the complex interlinked regulation and conditions that determine these two distinct metabolic states and govern the transition between them. In this review, we discuss these models with a focus on the mechanisms controlling intra- and extracellular changes between acidogenesis and solventogenesis. In particular, we critically evaluate underlying model assumptions and predictions in the light of current experimental knowledge. Towards this end, we briefly introduce key ideas and assumptions applied in the discussed modelling approaches, but waive a comprehensive mathematical presentation. We distinguish between structural and dynamical models, which will be discussed in their chronological order to illustrate how new biological information facilitates the ‘evolution’ of mathematical models. Mathematical models and their analysis have significantly contributed to our knowledge of ABE fermentation and the underlying regulatory network which spans all levels of biological organization. However, the ties between the different levels of cellular regulation are not well understood. Furthermore, contradictory experimental and theoretical results challenge our current notion of ABE metabolic network structure. Thus, clostridial ABE fermentation still poses theoretical as well as experimental challenges which are best approached in close collaboration between modellers and experimentalists

Biobutanol production from cellulosic and sugar-based feedstock from the corn plant

In this thesis, biobutanol production by biological fermentation was investigated from the corn plant, integrating two approaches. The first one was to utilize corn cobs, a cellulosic-based material. The second, using a new sugar-based material, sugarcorn juice. Utilizing suitable Clostridia strains for each substrate, these approaches converged into a biorefinery concept to produce renewable biofuels in Ontario, Canada. The corn cob pretreatment was carried out by a dilute acid method resulting in temperature as the variable with most significant effect towards glucose liberation. The enzymatic hydrolysis was performed utilizing a very low concentration of an enzymatic stock solution with approximately 44% of hydrolysis conversion. Biobutanol fermentation was pursued utilizing a Clostridium beijerinckii strain and cellulosic biobutanol was produced in a concentration of 4.42 g L-1 at 48h with 97% of reducing sugars utilization. Different ABE fermentations by Clostridium saccharobutylicum ATTC BAA-117 using glucose, fructose, sucrose, and a mix of them, resulted in butanol production as high as 12-14 g L-1. For the first time, sugarcorn juices from Canadian corn hybrids, were characterized and proven as a suitable medium for biobutanol production. Variation in sugar composition of sugarcorn juices across different hybrids and growth seasons were observed during this study, from 102 g L-1 to 145 g L-1, with fructose, glucose and sucrose accounting for about 80%. Clostridium beijerinckii 6422 produced 8.49 g L-1 of butanol over 257h of fermentation utilizing sugarcorn juice as substrate. It had a biphasic fermentation where acids accumulation happened at the beginning of fermentation. Interestingly, at the end of the fermentation butyric acid was reactivated and the butanol production shifted towards butyric acid production. Clostridium saccharobutylicum produced 11.05 g L-1 of butanol over 227 h of fermentation utilizing sugarcorn juice as substrate. Both strains could utilize sucrose, fructose and glucose concomitantly. There is enough evidence to agree that Clostridium saccharobutylicum has a PTS-sucrose system which allows the cell to transport sucrose inside the cell. The proposed Canadian sugarcorn (CANSUG) biorefinery can commercially generate biofuels and biochemicals while limiting wastes, offer environmental benefits to the energy sector, and strengthen the Canadian bio-economy

Metabolic engineering of acid formation in Clostridium acetobutylicum

During the last few decades, there has been an increasing search for alternative resources for the production of products traditionally derived from oil, such as plastics and transport fuels. This has been prompted by the finite nature of our oil reserves, the desire for energy security, and by concerns about anthropogenic global warming. Petrol and diesel are the two main fuel types for land based transportation and are currently derived from oil. Butanol, a four-carbon alcohol that can be produced by certain bacteria in a renewable way, can be used as a direct petrol replacement. It also has multiple applications as chemical intermediate and as a solvent. Although it is similar to ethanol it has superior properties with regard to energy density, vapour pressure, and water solubility when applied a biofuel. The acetone-butanol-ethanol (ABE) fermentation of sugars as carried out by various bacteria of the genus Clostridium has been widely applied in the first part of the 1900s as a commercial method to produce butanol and acetone. The two most used species have been Clostridium acetobutylicum and C. beijerinckii. Both produce not only solvents but also the unwanted acids acetate and butyrate. In the second part of the 20th century, the ABE-process became no longer economically competitive with the petrochemical process for the production of these solvents. But today’s high oil prices make the fermentation process interesting again, although there are still challenges that have to be tackled before the process can be re-commercialised. These include finding ways to make it possible to use cheap biomass feedstocks (such as lignocelluloses) as substrate rather than using traditional feedstocks such as starch and molasses, which are relatively expensive. In addition this replacement would avoid the food-versus-fuel dilemma. Another challenge is to improve butanol production, yield, and titre. The work described in this thesis focuses on the enhancing of butanol production and diminishing acid formation by C. acetobutylicum. A metabolic engineering approach was taken to reduce the number and amount of by-products in C. acetobutylicum fermentations. Production pathways of the acids acetate and butyrate were targeted, as we hypothesised that inhibiting acid formation would also prevent acetone production by C. acetobutylicum, resulting in only alcohols as the liquid fermentation products. To carry out our metabolic engineering work, we first developed an essential tool for gene disruption. During this work we studied storage conditions for electro-competent C. acetobutylicum cells, allowing for the batch preparation of these cells for later use for up to 54 months (Chapter 2 part 1). The principle on which it is based, exclusion of oxygen, suggests that it might also be applicable to the storage of other obligate anaerobes. The second part of Chapter 2 describes the adaptation of the TargeTron gene knock-out system for use in C. acetobutylicum. The TargeTron system uses a mobile group II intron that can be ‘retargeted’, i.e. reprogrammed, to insert into a specific site in the genome in a process called retrohoming. We targeted the acetate kinase (ack) gene and successful insertion of the intron was demonstrated using a PCR test. But only after the development of a colony PCR protocol for C. acetobutylicum as described in Chapter 4, we were able to apply our system and quickly detect pure mutants amongst the parental strain. Another research group also developed a clostridial version of the TargeTron system and called it ClosTron. The advantage of this system over the one we developed is that inserted intron copies carry an activated erythromycin resistance gene and can therefore easily be selected. In Chapter 3 we used this system to obtain an acetate kinase gene knockout, which was extensively characterised in pH‑controlled batch fermentations on two media; CGM and Clostridial Medium 1 (CM1). Enzyme assays showed a 98 % reduction in in vitro acetate kinase activity, however the mutant strain continued to produce wild-type levels of acetate in CGM which does not contain any added acetate. In CM1 that does contain acetate, acetate production could still be seen, but was severely reduced. These results suggest that alternative ways of acetate production may be active in C. acetobutylicum. The solvent production of the ack— strain was not significantly affected in CM1. When grown on CGM our wild-type strain produced large amounts of lactate and was therefore not suitable as a production medium. Interestingly our ack— mutant strain performed better. Subsequently we created a strain with an inactivated butyrate kinase gene termed BUK1KO, as described in Chapter 4. The phenotype of this strain was essentially that of an acetate-butanol producer. Analysis of the fermentation behaviour indicated that the strain never seemed to switch from an acidogenic to an solventogenic state, as the wild-type did. Furthermore, the growth on CM1 in batch culture demonstrated a strong influence of the pH on the fermentation behaviour. There was a good correlation between increasing fermentation pH and higher acetate levels within the pH range from 4.5 to 5.5, suggesting that the produced acetate levels might actually be the growth inhibiting compound. In addition, the mutant cells never produced the clostridial cell-types associated with spore formation. This is in line with the absence of a solventogenic switch. Also in parallel with the increasing fermentation pH was an increased acetoin accumulation with a maximum of 49 mM at pH 6.5 compared to 12 mM for the wild type under control conditions. Growth on CM1 without acetate at a pH of 5.5 resulted in a 21 % increase in butanol levels to 195 mM (14.5 g/L) compared to the wild type under its optimal conditions and 127 % under the same conditions. There was also a 60 % reduction in acetone levels and slightly increased ethanol levels. A subsequent inactivation of the acetate kinase gene in the buk1— negative background using our own TargeTron system (see Chapter 2) resulted in isolation of an ack— buk1— double mutant. Despite abolishment of both acetate kinase and butyrate kinase enzyme activity in vitro, the mutant continued to produce both acids. In CM1, acetate levels were severely reduced compared to the parenteral buk1— strain, but when acetate was removed from the medium, large amounts of acetate were produced again. This behaviour is reminiscent of the ack— mutant and supports the hypothesis that unknown alternative acid producing pathways or enzymes exist in C. acetobutylicum. Alcohol production was negatively affected as compared to the parental strain and acetone production was not eliminated. Also at certain pH‑levels acetoin production was even further increased to 100 mM, the highest reported value for this organism. In an alternative take on improving butanol production titre, we envisioned a homo-fermentative 2‑butanol strain. 2‑butanol is less toxic to the cell and should, in the proposed pathway, be produced redox-neutral from glucose. In addition it retains all the beneficial biofuel properties. As a first step towards this goal, we demonstrated in Chapter 5 that an alcohol dehydrogenase from Clostridium beijerinckii, over-expressed in C. acetobutylicum, can accept natively produced d‑ and l‑acetoin as its substrate and reduce it to d‑ and meso‑2,3‑butanediol. In addition we showed that our C. acetobutylicum WUR strain already produces small amounts (approximately 3 mM) of meso‑2,3‑butanediol through an unknown pathway, most likely from d‑acetoin. No production of meso‑2,3‑butanediol was observed for the ATCC 824 strain. Completion of the pathway requires a dehydratase and a secondary-alcoholdehydrogenase to produce methyl-ethyl ketone and 2‑butanol respectively. In the general discussion (Chapter 6) the results described in this thesis were put into perspective, and the existence of an alternative acid pathway in C. acetobutylicum is suggested. Furthermore the disadvantages and advantages of C. acetobutylicum as a butanol production platform are discussed together with developments of butanol production in heterologous hosts.</p

Automation on the generation of genome scale metabolic models

Background: Nowadays, the reconstruction of genome scale metabolic models is a non-automatized and interactive process based on decision taking. This lengthy process usually requires a full year of one person's work in order to satisfactory collect, analyze and validate the list of all metabolic reactions present in a specific organism. In order to write this list, one manually has to go through a huge amount of genomic, metabolomic and physiological information. Currently, there is no optimal algorithm that allows one to automatically go through all this information and generate the models taking into account probabilistic criteria of unicity and completeness that a biologist would consider. Results: This work presents the automation of a methodology for the reconstruction of genome scale metabolic models for any organism. The methodology that follows is the automatized version of the steps implemented manually for the reconstruction of the genome scale metabolic model of a photosynthetic organism, {\it Synechocystis sp. PCC6803}. The steps for the reconstruction are implemented in a computational platform (COPABI) that generates the models from the probabilistic algorithms that have been developed. Conclusions: For validation of the developed algorithm robustness, the metabolic models of several organisms generated by the platform have been studied together with published models that have been manually curated. Network properties of the models like connectivity and average shortest mean path of the different models have been compared and analyzed.Comment: 24 pages, 2 figures, 2 table

Estrategia de modificación metabólica de una cepa nativa de Clostridium para la producción De 1,3-Propanodiol a partir de Glicerol

El incremento de la producción de fuentes energéticas alternativas como biodiesel ha conllevado a estudiar la posibilidad de establecer biorrefinerías donde se haga uso del subproducto glicerol sin purificar como sustrato para la producción de 1,3-Propanodiol (1,3-PD) por parte de microorganismos del género Clostridium. Con el propósito de establecer una estrategia de modificación genética a partir del análisis del transcriptoma de Clostridium sp. IBUN13A para la producción de 1,3-PD se obtuvo inicialmente su genoma, lo que otorgó un panorama global del potencial metabólico de la cepa, así como la base de la construcción de un modelo metabólico. Así mismo se realizó un estudio de genómica comparativa entre 32 cepas del género, mostrando que este tiene una amplia plasticidad génica. Tomando en cuenta la tendencia actual de comprender globalmente la información que se puede obtener a partir de técnicas moleculares de punta y generar modelos predictivos de los organismos vivos a partir de la biología de sistemas, se utilizó la red metabólica obtenida previamente dentro del grupo de investigación para implementar estrategias de manipulación in silico de triples mutantes, que nos permitieron evaluar la viabilidad de modificar de forma racional al microorganismo y para lo que no se encontraron incrementos en el rendimiento molar del diol de más de 2%. De igual forma se establecieron las diferencias en el perfil transcriptómico global de la cepa en cultivo con glicerol respecto a glucosa, lo cual es fuente de información sobre la fisiología de esta bacteria y sobre posibles blancos de manipulación genética.Abstract: The increase in the production of alternative energy sources such as biodiesel, has encouraged the study about the possibility of establishing biorefineries where glycerol (byproduct of this process) can be used without purifying as a substrate to produce 1,3-propanediol (1,3-PD) by microorganisms of the genus Clostridium. In order to establish a genetic modification strategy based on the analysis of the transcriptome of Clostridium sp. IBUN13A for producing 1,3-PD, at first its genome was obtained, which gave an overview of the metabolic potential of the strain, as well as the basis of the construction of a metabolic model. Likewise, a study of comparative genomics was carried out among 32 strains of the genus, showing that it has a broad genetic plasticity. Taking into account the current trend of globally understanding the information that can be obtained from cutting-edge molecular techniques and generating predictive models of living organisms from Systems Biology, we used the metabolic network previously obtained within the research group to implement strategies of in silico manipulation of triple mutants, which allowed us to evaluate the feasibility of modifying the microorganism rationally and for which no were found increases in the molar yield of the alcohol of more than 2%. Also, the differences in the global transcriptomic profile of the strain cultured with glycerol were established with respect to glucose, which is a source of information about the physiology of this strain and possible targets of genetic manipulation.Doctorad

Exploring complex cellular phenotypes and model-guided strain design with a novel genome-scale metabolic model of Clostridium thermocellum DSM 1313 implementing an adjustable cellulosome

Additional file 1. Model structure and biomass term calculations